Although many biochemical tests are available for identifying micro-organisms in the Enterobacteriaceae family, differentiation of organisms such as Arizona or Citrobacter from Salmonella, and Pseudomonas cepacia or P. maltophilia from other pseudomonads has always been difficult. Recently, the o-nitrophenyl-.beta.-D-galactopyranoside test has proven useful in differentiating the above-mentioned organisms. This test detects .beta.-galactosidase-producing organisms by the bacterial hydrolysis of the substrate, o-nitrophenyl-.beta.-D-galactopyranoside. This last mentioned o-nitrophenyl-.beta.-D-galactopyranoside, commonly known as "ONPG, is an artificial substrate which is initially colorless in solution. Hydrolysis of the substrate by bacterially-produced .beta.-galactosidase enzyme, releases free o-nitrophenol, which is yellow under alkaline conditions. Thus, a convenient method for detecting .beta.-galactosidase activity and the micro-organisms which produce this enzyme, is provided.
The ONPG test has been the subject of a number of studies: Lederberg, J.,J. Bact. 60: 381-392 (1950); Lowe, G.H., J. Med. Technol. 19: 21-25 (1962); Pickett, M.J. et al., Appl. Microbiol. 14: 178-182 (1966); and Sonnenwirth, A.C., Chapter 62, Gradwohl's Clinical Laboratory Methods and Diagnosis, 7th Ed., 1970, pages 1111-1112. In all of the above studies, the ONPG substrate is prepared in a buffer solution at the time the test with an unknown organism is to be run. Pre-prepared substrate solutions of suitable sensitivity and stability have not been reported. Comparative studies on the use of an ONPG diagnostic test strip are reported by Rosner, R. in Appl. Microbiol. 26: 890-893 (December 1973). Results with the ONPG test strip were favorable, according to the Rosner studies, but neither the composition, configuration or method of preparing the test strip are disclosed in this paper. The ONPG test strip used in the Rosner studies is, in fact, an early attempt by the inventors of the diagnostic test system of this invention to prepare a stable, sensitive, product which would accurately detect .beta.-galactosidase-producing organisms. Unfortunately, the stability of the ONPG test strip studied by Rosner was so short-lived that neither sensitivity of the test product nor the accuracy of the results could be guaranteed should commercial development be undertaken. A pre-prepared diagnostic test system must be able to withstand the manufacturing, shipping and storage conditions normally encountered to be considered suitable for marketing. Such a product must provide reliable, reproducible test results. Thus, while some progress has been made, there is still a need for a pre-prepared, stable, sensitive ONPG test system which can be used for the rapid identification of micro-organisms which produce .beta.-galactosidase enzymes.